1 Carbonic anhydrase is involved in calcification by the benthic foraminifer Amphistegina 2 lessonii

3

4 Siham de Goeyse1, Alice E. Webb1, Gert-Jan Reichart1,2, Lennart J. de Nooijer1 5 6 1 Department of Ocean Systems, NIOZ Royal Netherlands Institute for Sea Research and Utrecht University, Texel, 7 Netherlands 8 2 Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Utrecht, Netherlands 9 10 *corresponding author: [email protected]

11 Key words: Foraminifera, calcification, symbiont, photosynthesis, carbonic anhydrase

12

13 Abstract

14 Marine calcification is an important component of the global carbon cycle. The mechanism by which some 15 organisms take up inorganic carbon for the production of their shells or skeletons, however, remains only partly 16 known. Although foraminifera are responsible for a large part of the global calcium carbonate production, the 17 process by which they concentrate inorganic carbon is debated. Some evidence suggests that seawater is taken up 18 by vacuolization and participates relatively unaltered in the process of calcification, whereas other results suggest 19 the involvement of transmembrane transport and the activity of enzymes like carbonic anhydrase. Here, we tested 20 whether inorganic carbon uptake relies on the activity of carbonic anhydrase using incubation experiments with 21 the perforate, large benthic, symbiont-bearing foraminifer Amphistegina lessonii. Calcification rates, determined 22 by the alkalinity anomaly method, showed that inhibition of carbonic anhydrase by acetazolamide (AZ) stopped 23 most of the calcification process. Inhibition of photosynthesis by either 3-(3,4-Dichlorophenyl)-1,1-dimethylurea 24 (DCMU) or by incubating the foraminifera in the dark, also decreased calcification rates, but to a lesser degree 25 than with AZ. Results from this study show that carbonic anhydrase plays a key role in biomineralization of 26 Amphistegina lessonii and indicates that calcification of those perforate, large benthic foraminifera might, to a 27 certain extent, benefit from the extra DIC which causes ocean acidification.

28 1 Introduction

rd 29 Fossil fuel burning and land use changes have been steadily increasing atmospheric CO2 levels. About 1/3 of the 30 added carbon has been taken up by the ocean (Sabine and Tanhua, 2010) and the resulting increase in seawater 31 dissolved carbon dioxide and associated acidification are lowering the saturation state of sea water with respect to 32 calcite and hence likely affects marine calcifiers. Even a modest impact on the production of carbonate shells and 33 skeletons may have important consequences for the global carbon cycle. Foraminifera are responsible for almost 34 25% of the total marine calcium carbonate production (Langer, 2008) and their response to ongoing acidification

35 is therefore important to predict future marine inorganic carbon cycling. Despite its relevance for future CO2

36 scenarios, it is still unclear how increased pCO2 in seawater will affect foraminiferal calcification. Previous 37 research has shown discrepancies in their results: in some cases a higher pCO2 increased the growth rate of benthic 38 foraminifera, while in other cases calcification decreased or halted (Haynert et al., 2014; Hikami et al., 2011).

39 Addition of CO2 to sea water not only reduces saturation state with respect to calcite but also increases the total - 40 dissolved inorganic carbon (DIC) concentration. At surface seawater pH, the dominant DIC species is HCO3 and 41 many marine calcifyers are shown to employ transmembrane bicarbonate ion transporters (e.g. coccolithophores 42 (Brownlee et al., 2015; MacKinder et al., 2011); scleractinian corals (Cai et al., 2016; Giri et al., 2019; Zoccola et 43 al., 2015)), which may also be the case for foraminifera. If so, ocean acidification would be detrimental as this - 44 shifts the carbonate system from HCO3 to CO2. Alternatively, CO2 may be the inorganic carbon source of choice 45 for benthic foraminifera, as it diffuses relatively easily through lipid membranes. The latter uptake mechanism

46 would facilitate foraminiferal calcification as ongoing CO2 dissolution increases total DIC and hence the 47 availability of building blocks for chamber formation. Since this uptake mechanism is crucial for calcification in 48 a rapidly changing ocean and because it is essentially unknown how foraminifera take up inorganic carbon, it 49 remains difficult to predict the reaction of foraminifera to ongoing environmental change. It was recently suggested

50 that CO2 uptake by benthic foraminifera is achieved through proton pumping (Glas et al., 2012; Toyofuku et al.,

51 2017). The outward proton flux increases the pCO2 directly outside the site of calcification (SOC) through 52 conversion of bicarbonate into carbon dioxide. The elevated pH at the foraminifers’ site of calcification (Bentov

53 et al., 2009; de Nooijer et al., 2009) and reduced pH outside the cell thus results in a strong inward-outward pCO2

54 gradient, promoting inward CO2 diffusion. If calcification in foraminifera relies on this inward CO2 diffusion, the - 55 conversion from HCO3 outside the test may be a limiting step for ongoing calcite precipitation. This process may 56 be catalyzed by an enzymatic conversion by carbonic anhydrase (CA), which is present in many prokaryotes and 57 virtually all eukaryotes (Hewett-Emmett and Tashian, 1996; Lionetto et al., 2016). This enzyme is essential in 58 calcification in many organisms, including corals, sponges and coccolithophores (Bertucci et al., 2013; Medaković, 59 2000; Müller et al., 2013; Le Roy et al., 2014; Wang et al., 2017). Also for foraminiferal calcification it has been 60 hypothesized that CA is used to enhance inorganic carbon uptake. Indirect evidence for such a role in calcification 61 comes from the observed slope between the carbon and oxygen isotopes (Chen et al., 2018), but direct evidence 62 is, however, still missing. 63 64 To test whether carbonic anhydrase is involved in biomineralization of perforate, benthic foraminifera we 65 incubated calcifying specimens of Amphistegina lessonii with acetazolamide (AZ), a membrane-impermeable 66 inhibitor of this enzyme (Elzenga et al., 2000; Moroney et al., 1985). Calcification and respiration were determined 67 by measuring changes in alkalinity and DIC of the incubated seawater over the course of the experiment. An 68 additional experiment was conducted in parallel to test whether CA is directly involved in perforate foraminiferal 69 calcification or that the effect is indirect via photosynthesis. The latter would imply that CA drives photosynthesis 70 by the symbionts and that observed effects would be due to reduced photosynthesis impairing calcification through 71 reduced energy transfer from the symbionts to the foraminifer.

72 2 Material and methods

73 2.1 Foraminifera and incubations 74 Surface sediments were collected from the Indo-Pacific Coral reef aquarium in Burgers’ Zoo (Arnhem, the 75 Netherlands; Ernst et al., 2011). The sediments were kept at 24 ⁰C, with a day/night cycle of 12h/12h. Living 76 specimens of Amphistegina lessonii showing a dark cytoplasm and pseudopodial activity were manually selected, 77 using a fine brush under a stereomicroscope and transferred to Petri dishes. They were fed with freeze-dried 78 Dunaliella salina and incubated in North Atlantic sea water (salinity: 36). After a week, viable specimens were 79 collected and divided over eight experimental conditions, each of them consisting of three groups (Fig. 1). Each 80 group consisted of 40-60 specimens with a similar size distribution (initial diameter: 140 to 1200 µm, shown in 81 S1). Foraminifera were placed in air-tight glass vials of 80 ml (24°C, 12h day-light cycle) for 5 days. Illumination 82 was approximately 180 µmol photons m−2 s−1, during the 12h of light. 83 84 In the first experiment, the impact of acetazolamide (AZ) on calcification was tested. A stock solution was prepared 85 by dissolving AZ (Sigma-Aldrich) in dimethyl sulfoxide (DMSO; 0.05% v/v) at a final concentration of 90 mM. 86 It has been shown that DMSO at concentrations of 10-20% v/v does not impair calcification (Moya et al., 2008), 87 so that the effect of this solvent is not reported here separately. The AZ stock solution was diluted with seawater 88 from North Atlantic to achieve AZ concentrations of 4, 8 and 16 µM, which were used to incubate the foraminifera 89 in. In a second experiment, inhibition of photosynthesis was tested by 1) addition of 3-(3,4-Dichlorophenyl)-1,1- 90 dimethylure (DCMU ; Tóth et al., 2005; Velthuys, 1981) and 2) darkness. DCMU was added to seawater at a final 91 concentration of 6 µM, whereas covering the vials with aluminum foil prevented light-dependent reaction and 92 hence photosynthesis in a second set of incubations (Fig. 1). 93 94 95 2.2 Alkalinity, DIC and nutrient analysis 96 97 To quantify calcification and respiration, total alkalinity (TA) and the concentration of dissolved inorganic carbon 98 [DIC] were determined at the beginning and end of every incubation. This method was chosen above other growth 99 method measurements such as sample weighing or counting chamber addition as it allows a quantification of the 100 amount of calcite formed during the actual experiment. Total alkalinity was analyzed immediately at the end of 101 each experiment, whereas subsamples to determine nutrient concentrations and DIC analyses were stored at -20°C 102 (nutrients) and 4°C (DIC). The samples for DIC analyses were poisoned with mercury chloride (DIC) until 103 analysis. These samples first passed a 0.2 µm syringe filter. 104 105 Alkalinity measurements were performed using an Automated Spectrophotometric Alkalinity System (ASAS), as 106 described by Liu et al. (2015). Briefly, 60 mL of seawater are placed in a borosilicate vial and automatically titrated 107 with a solution of 0.1 M HCl. Before the start of the titration, 45 microliters of bromocresol purple (10 mmol/L) 108 was added to the seawater and pH changes were followed by spectrophotometry. Certified reference material 109 (CRM; Dr. Dickson, Scripps Institution of Oceanography) was analyzed at the beginning of every series (5-10

110 samples) of measurements. Reproducibility of the obtained TA was ~3 µmol/kg (SD), based on 50 measurements 111 of untreated seawater. 112 113 Nutrient samples were analysed on a QuAAtro continuous flow analyzer (SEAL Analytical, GmbH, Norderstedt, 114 Germany) following GO-SHIP protocol (Hydes et al., 2010). DIC was measured on an autoanalyzer TRAACS 115 800 spectrophotometric system as described in Stoll et al. (2001). 116 117 2.3 Calcification rate 118 119 Changes in DIC and alkalinity between start and end of the experiments were used to calculate the net respiration 120 and calcification (Fig. 2). Total measured alkalinity is defined as the contribution of the following anions: − 2− − 3− 2− - + + 121 TAmeasured = [HCO3 ] + 2[CO3 ] + [OH ] + 3[PO4 ] + [HPO4 ] + [NO3 ] - [H ] -[NO4 ] (1) 122 Concentrations of boron and silicon were neglected as the first one is constant and the second one is present at a 123 low abundance. In order to account for the alkalinity change related to the inorganic carbon system only, we 124 subtracted the combined concentrations of the nutrients from the measured alkalinity so that the observed alkalinity 125 over time is defined as: − 2− − + 126 TA = [HCO3 ] + 2[CO3 ] + [OH ] -[H ] (2) 127

128 Respnet is defined as the difference between respiration and photosynthesis. Here, we consider the respiration of 129 the holobiont (foraminifera and its symbionts), which is calculated by: 130 = /2 (3)

131 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅 𝑛𝑛𝑛𝑛𝑛𝑛 ∆ 𝐷𝐷𝐷𝐷𝐷𝐷 − ∆ 𝑇𝑇𝐴𝐴 132

133 Since other processes, e.g. respiration by bacteria, may affect the TA and [DIC] during the incubations, vials were 134 carefully checked for the presence of biofilms. There was no sign of such activity in any of the treatments, so

135 changes in TA and [DIC] are attributed to the foraminifera and their symbionts.

136 3 Results

137 3.1 Carbonic anhydrase inhibition 138 -1 -1 139 Without acetazolamide, TA decreased on average by 53 µmol.kg and DIC by 38 µmol.L during the incubation 140 (table 1). This corresponds to 2.74 g/L of precipitated calcite. Contrastingly, when the seawater contained 141 acetazolamide (even at the lowest concentration of 4µM), alkalinity and DIC did not change or decreased only

142 marginally during the incubation (less than 0.4 g/L of calcite precipitated). When comparing the changes in TA 143 and DIC between treatments, calcification is minimized by the AZ and net respiration slightly increases (Fig. 3). 144 The concentration of AZ has no discernible effect on the magnitude of changes in calcification/ respiration. 145 146 The number of chambers added by the foraminifera shows that the average number of chambers added decreases 147 after addition of AZ (table 3). Whereas many specimens in the control vials added 2 chambers, almost all 148 calcification after addition of AZ resulted in the addition of only one chamber. 149 150 3.2 Photosynthesis inhibition 151 152 When photosynthesis was not impaired (light control), alkalinity decreased within the vials by 70 μmol·L−1 and −1 153 DIC increased by 21 μmol·L (table 2). Given the relative standard deviations, this is similar to the changes in TA 154 and DIC in the control vials for the AZ-experiments. These changes correspond to approximatively 3.75 g·L-1 of 155 precipitated calcite. In contrast, when foraminifera were cultivated in the dark or in presence of the photosynthesis 156 inhibitor DCMU, DIC increased by 16 and 42 μmol·L−1, respectively whereas the total alkalinity decrease was 157 only 19 and 11 μmol·L−1, respectively, which corresponds to less than a third of the amount of calcite precipitated

158 when photosynthesis was not hampered (Fig. 4). Changes in DIC/ TA are also reflected in the number of chambers 159 added to the incubated foraminifera: with DCMU or AZ added and in the dark, specimens added less chambers 160 than the control group (table 3). Some of the smaller specimens incubated during the experiment were not retrieved 161 from the vial, explaining the missing specimens (table 3). The foraminifera incubated with an inhibitor have more 162 broken chambers than the others. 163

164 4. Discussion

165 4.1 Growth rates and the effect of AZ 166 In the control experiments (incubations with unaltered seawater), foraminiferal calcification resulted in a decrease 167 in alkalinity of the culture media by approximately 53 μmol·L−1 over a period of 5 days (table 1). On average, this 168 equals a growth rate of 1.0 μg·Ind.−1·day−1, which is low when compared to some previously reported rates for 169 foraminiferal calcification (~6-60 μg·Ind.−1·day−1; (Evans et al., 2018; Glas et al., 2012; Keul et al., 2013). These 170 studies, however, all used different species than the one incubated here. Previous research using Amphistegina spp. 171 reported growth rates of 3-9 and 2.6-4 μg·Ind.−1·day−1 (ter Kuile and Erez, 1984; Ter Kuile and Erez, 1987), 172 respectively, while Hallock et al. (1986) reported rates of 0.3-6.6 μg·Ind.−1·day−1 depending on the light intensity. 173 Segev and Erez (2006) reported growth rates similar to those observed in our study (0.53-1.0 μg·Ind.−1·day−1), 174 based on changes in dry weight. The growth rates reported here fall in the lower range of those previously reported, 175 which may be due to the average size of our specimens, the used light intensity and/ or the short duration of our 176 experiment. 177 178 Addition of AZ caused a 20 fold decrease in calcification rates (Fig. 3), while increasing net respiration. The 179 concentration of the inhibitor (4-16 µM) did not affect the magnitude by which net calcification decreased, nor 180 does it appear to affect the increase in net respiration (Fig. 3). The accompanying decrease in the number of 181 chambers added per specimen (table 3), suggests that AZ did not decrease the survival rates of the incubated 182 specimens, but affected the rate of chamber addition in all specimens equally. The inhibition of calcification caused 183 by AZ suggests that carbonic anhydrase plays a crucial role in perforate foraminiferal biomineralization. With the 184 inhibitor present, specimens produced little to no calcite (Fig. 3), indicating that either biomineralization relies on 185 CA, or is negatively impacted through an effect of CA on photosynthesis. Whether calcification depends directly 186 on extracellular carbonic anhydrase (eCA) or that calcification depends on photosynthesis and thereby indirectly 187 on CA, can be inferred from comparing the two sets of experiments (Fig. 1). 188 189 4.2 Effect of photosynthesis on calcification 190 The inhibition of photosynthesis with DCMU and darkness decreases calcification comparably (Fig. 3). 191 Simultaneously, net respiration increases after addition of DCMU, and so does blocking light (Fig. 4). The 192 similarity in the effect of darkness and DCMU indicates that photosynthesis has an effect on calcification in these 193 perforate foraminifera. It was previously suggested that light, irrespective of photosynthesis, enhances calcification 194 in foraminifera (Erez, 2003). Since the latter study used the planktonic, low-Mg calcite Globigerinoides sacculifer, 195 the discrepancy between results may be caused by differences in the process involved in calcification between 196 these species. For example, it has been suggested that calcification may involve seawater transport (Erez, 2003; 197 Segev and Erez, 2006) as well as transmembrane transport (Nehrke et al., 2013; Toyofuku et al., 2017), of which 198 the relative contribution may vary between groups of foraminifera. 199 Foraminiferal calcification and endosymbiont photosynthesis both require inorganic carbon. Therefore, it seems 200 reasonable to suggest that those two mechanisms are competing with each other for inorganic carbon, as was 201 shown by Ter Kuile et al. (1989b, 1989a). However, our results show that preventing photosynthesis by the 202 symbionts actually decreases foraminiferal calcification. This implies that benefits from photosynthesis overcomes 203 an eventual competition with calcification, which is in agreement with results from Duguay (1983) and Hallock 204 (1981) who showed that both calcium- and inorganic carbon uptake into the cell is enhanced by light. As the 205 foraminifera were in the dark 12h hours a day it might also be that DIC is shared over time, being used for

206 calcification during the dark phase and CO2 being used for calcification during the light phase. 207 It was shown that photosynthetic symbionts provide energy to their foraminiferal hosts (Lee, 2001) and that 208 calcification in some foraminifera is enhanced by the photosymbiont’s activity (e.g. Hallock, 2000; Stuhr et al., 209 2018). This was for example seen already by Muller (1978), reporting increased carbon fixation by the foraminifer

210 A. lessonii in the light compared to uptake of carbon in the dark. A positive effect of higher CO2 level on 211 calcification through enhanced photosynthesis is known as “fertilization effect” (Ries et al., 2009). A positive 212 effect of photosynthesis on calcification has been observed previously for other marine calcifyers as well. For

213 example, in coccolithophores, decreasing CO2 can hamper calcification through reduced photosynthesis 214 (Mackinder et al., 2010). Utilization of photosynthate as an organic template for calcification may explain this 215 observation. We here hypothesize that a similar effect may explain decreased calcification in foraminifera as a 216 consequence of inhibited photosynthesis (Fig. 3), as hypothesized by Toler and Hallock (1998). If so, the type of 217 organic molecules produced by the foraminifer’s endosymbionts and their fluxes will need to be assessed to test 218 the extent of the dependency of calcification on photosynthesis. However, it has been shown that symbiotic 219 dinoflagellates can trigger the activity of carbonic anhydrase from their host organisms (giants clams and sea 220 anemones) (Leggat et al., 2003; Weis, 1991; Weis and Reynolds, 2002; Yellowlees et al., 2008), thereby explaining 221 how photosynthesis enhances calcification. Alternatively, increased activity of CA in the symbiont may also 222 promote the flux of products to the host and thereby promote calcification indirectly. Since there are many 223 (perforate) foraminiferal species that do not have photosynthetic symbionts, the effect of inhibiting CA in these 224 species may provide additional information on the role played by CA in calcification. 225 226 4.3 Role of CA in calcification 227 In calcifyers other than foraminifera, carbonic anhydrase plays a direct role in calcification. For example, in giant 228 clams (Chew et al., 2019), gastropods (Le Roy et al., 2012) and oysters (Wang et al., 2017), CA helps to concentrate 229 inorganic carbon in the fluid from which calcium carbonate precipitates. In scleractinian corals, CA promotes

230 conversion of metabolic CO2 into bicarbonate after the carbon dioxide diffused into the sub-calicoblastic space 231 (Bertucci et al., 2013). Although the inorganic carbon would take the same route in absence of CA, the hydration

232 of CO2 is relatively slow and ion fluxes and calcification rates would be a fraction what they are with the catalytic 233 activity of CA. This role of CA fits with the localization of (membrane-bound) CA observed at the walls of the 234 calicoblastic cells by immunolabelling (Moya et al., 2008). In addition, by facilitating an inward flux of inorganic 235 carbon, involvement of CA can explain the co-variation of oxygen and carbon isotopes in coral aragonite (Chen 236 et al., 2018; Uchikawa and Zeebe, 2012).

237 In larger benthic foraminifera, CA likely plays different roles: it helps concentrating CO2 by the symbionts and 238 aids foraminiferal calcification. It is also likely that cytoplasmic CAs -involved for instance in intracellular pH 239 regulation- also affect calcification. The molecular types of CA that are involved and their precise location still 240 remain to be investigated within the larger benthic foraminifera. In addition, the type of symbionts or their absence, 241 may affect inorganic carbon uptake, so that the result obtained here may only partially apply to foraminifera in 242 general. Analogous to other calcifying organisms and based on existing models of foraminiferal calcification, we - 243 hypothesize that extracellular CA helps to convert HCO3 into CO2 directly outside the calcifying chamber. This

244 would help to further increase the pCO2 outside the foraminifer in addition to the shift in inorganic carbon 245 chemistry resulting from active proton pumping and subsequent low pH (Glas et al., 2012; de Nooijer et al., 2009; 246 Toyofuku et al., 2017). Although not directly targeted by our experimental approach, as the inhibitor we used is 247 membrane impermeable, it is likely that a form of CA within the calcifying fluid increases the rate by which the

248 diffused CO2 is converted into bicarbonate. 249 The involvement of extracellular CA in calcification may explain why perforate foraminifera can be relatively 250 resilient to ocean acidification. It also remains to be investigated whether Tubothalamea, who produce their calcite 251 in a fundamentally different way (Mikhalevich, 2013; Pawlowski et al., 2013), use CA similarly. If they rely on - 252 CA for conversion of HCO3 to CO2 and take up inorganic carbon by diffusion of CO2, additional dissolved

253 atmospheric CO2 may be beneficial for calcification in foraminifera. If they exclusively rely on bicarbonate ions, - 254 a reduction in pH would lower the [HCO3 ] and thereby hamper calcification. Manipulation of the inorganic carbon 255 speciation in relation to calcification and the aid of enzymes therein, will allow predicting rates of calcification as 256 a function of ongoing ocean acidification.

257 5 Conclusions

258 The alkalinity anomaly method allowed us to quantify growth rates in incubation experiments, equalling addition 259 of 1 µg/individual/day. Calcification and photosynthesis in the benthic foraminifer Amphistegina lessonii and its 260 symbionts both depend on carbonic anhydrase (CA) as shown after inhibition by acetazolamide (AZ). Since the 261 inhibitor is membrane-impermeable, the CA may well be localized at the outside of the foraminifer’s cell 262 membrane. Our results also show that inhibiting photosynthesis by DCMU or incubation in darkness reduce 263 calcification similarly. This suggests that not light, but photosynthesis itself promotes calcification in symbiont- 264 bearing perforate foraminifera. We also suggest that CA plays a role in concentrating inorganic carbon for 265 calcification, possibly by promoting conversion of bicarbonate into carbon dioxide outside the foraminifer.

266 Data availability

267 The data on which this publication is based can be found through the following DOI: 10.4121/uuid:afcdcdc1-2591- 268 4822-bade-806119cdd724 269 270 Authors contribution: 271 SdG and LJdN designed the experiment and SdG carried it out. SdG and AEW analysed the seawater inorganic 272 chemistry. SdG and LJdN analysed the data and prepared the manuscript with contributions from all co-authors. 273

274 Competing interests

275 The authors declare they have no conflict of interest

276 Acknowledgments

277 We would like to thank Karel Bakker for DIC measurements. We kindly thank Max Janse (Burgers’ Zoo, Arnhem) 278 for providing stock specimens of Amphistegina lessonii and Kirsten Kooijmans and Michele Grego (NIOZ) for 279 providing cultures of Dunaliella salina. This study was also carried out under the program of the Netherlands Earth 280 System Science Centre (NESSC; 024.002.001), financially supported by the Dutch Ministry of Education, Culture 281 and Science (OCW). LdN acknowledges financial support from the NWO open competition program (Pasodoble 282 Grant). We thank Takashi Toyofuku, the three anonymous reviewers and the editors for their helpful comments 283 and all subsequent improvements.

284 References

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437

438 Figure 1: 59 specimens were placed in one culture vial, with three replicate vials for each concentration of acetazolamide 439 (upper row). Similarly, 42 specimens were incubated under light, in the dark and with the inhibitor DCMU (lower row). 440 441 Figure 2: Calcification and net respiration of foraminifera deduced from changes in DIC and total alkalinity over time. 442 The blue vectors show the impact of photosynthesis and respiration (impacting DIC), the red arrow show the impact of 443 calcification and calcite dissolution (impacting both DIC and TA in a 1:2 ratio). Observed changes for each incubation 444 should be decomposed into two vectors: a contribution of calcification (dashed red arrow) and the net effect of 445 respiration and photosynthesis (dashed blue arrow). Approach is indicated here for a hypothetical incubation). 446 447 448 Figure 3: Changes in total alkalinity versus DIC for all concentrations of acetazolamide (AZ) used. Every black circle 449 represents the average change in DIC-TA for one triplicate of incubations. The three grey circles show the measured 450 DIC/ TA combination for each of the triplicate measurements within the control treatment. For the three additions of 451 AZ, replicates never differed more than 8 µmol/kg from the average for DIC and never more than 5 µmol/kg from the 452 average for TA. Arrows show the calcification (red) and net respiration (blue) effects. 453 454 455 Figure 4: Changes in total alkalinity versus that in DIC for incubations in light-dark alternation (control), in the dark 456 and with the photosynthetic inhibitor DCMU. Every black circle represents the average change in TA and DIC between 457 the initial and the final values for each triplicate. The three grey circles show the measured DIC/ TA combination for 458 each of the triplicate measurements within every of the three treatments. For the ‘dark’ and ‘DCMU’ treatments, the 459 individual DIC/TA combinations are connected to the average value. Arrows show the calcification (red) and net 460 respiration (blue) effects. 461 462

463 464 [AZ] Initial Initial Initial Initial (µM) TA Δ TA DIC Δ DIC Vial TA Δ TA DIC Δ DIC 0 2284 - 53± 8 2110 -38 ± 9 control 2280 -70 ±7 2115 -21 ± 9 4 2285 -7 ± 1 2105 -2 ±2 DCMU 2286 -22 ±9 2091 42 ±14 8 2285 -5 ± 1 2105 3 ± 7 dark 2280 -19 ±6 2115 16 ±5 16 2292 -2 ± 4 2109 -3 ±6

Table 1: Total alkalinity and DIC changes for every Table 1 : Total alkalinity and DIC changes triplicate. Confidence interval: 1 STD (taking for every triplicate. Confidence interval: 1 biological variability into account) STD (taking biological variability into account)

Total no of Number of specimens that added: Experiment specimens 1 chamber 2 chambers 3 chambers 4 chambers incubated AZ, 0 µM 80 25 19 1 1 AZ, 4 µM 100 17 4 0 0 AZ, 8 µM 123 15 2 0 0 AZ, 16 µM 135 6 0 0 0 control, light 123 40 25 1 0 DCMU 115 16 1 0 0 dark 122 18 0 0 0

Table 3: Number of chambers added per specimen for each treatment